The distribution of tenascin was examined in the lesioned adult rat optic nerve and central nervous system (CNS) non-neuronal cells in vitro, by means of a double immunofluorescence technique. Tenascin-like immunoreactivity is localized to the leptomeninges and astrocytes that border the site of optic nerve transection. Anti-tenascin labeling was observed as early as 24 hours after transection, when it appeared as a fine interface between leptomeninges and neural tissue. The anti-tenascin labeling increased in the cells at this border zone during the next 2 weeks, and disappeared 18-21 days after transection. In vitro studies further confirmed that both astrocytes and leptomeningeal cells express tenascin as detected by immunofluorescence labeling with anti-tenascin antibodies. However, the pattern of immunolabeling associated with the two cell types differed. Astrocytes showed exclusively punctate labeling of the cell surface, while leptomeningeal cells showed mainly coarse, fibrillary, matrix-like deposits. Astrocytes and leptomeningeal cells remained segregated when cocultured. In these cultures, an increased amount of the fibrillary, matrix-like deposits of tenascin was also observed in the region of the interface between astrocytes and leptomeningeal cells when these two cell types contact each other. Given the antiadhesive and antispreading properties of tenascin, these in vivo and in vitro results suggest that tenascin might play a role in the initial segregation of leptomeningeal cells from neural tissue at the site of CNS trauma during the first 2 weeks after injury, i.e., prior to the formation of a fully differentiated glia limitans. Therefore, tenascin may influence the early stages in the formation of the glia limitans, and thus prevent the indiscriminate migration of leptomeningeal cells into CNS tissue after injury.